Project Summary Mycobacterium tuberculosis (Mtb) infects 2 billion people worldwide, and kills more people every year than any other single pathogen. Most people infected with Mtb are able to contain infection for their lifetimes, suggesting the existence of immune mechanisms that can successfully control infection. Identifying these mechanisms is crucial for the development of therapeutics that can bolster immunity in patients with insufficient immunity. Macrophages serve the dual role as both the host cell for Mtb infection, and the cell that is primarily responsible for controlling infection by activating microbicidal mechanisms that effectively kill bacteria. In addition, macrophages influence the inflammatory response to infection by producing both pro-and anti- inflammatory factors. The long-term goal of this project is to understand how macrophage metabolism influences both antimicrobial activity and the regulation of inflammation. Our previous work centered around activation of macrophages by IFN-?, a cytokine that is critical for immune control of Mtb. We found that an immuno-metabolic loop linking aerobic glycolysis, nitric oxide, and the transcription factor HIF-1? is crucial for both antimicrobial control and regulating the balance of inflammation in macrophages infected ex vivo. In addition, we demonstrated that HIF-1? in macrophages in essential for control of infection in mice. However, it remains unclear whether the importance of HIF-1? and nitric oxide in vivo result from cell intrinsic control by macrophages, regulation of inflammation, or both. Furthermore, recent data from several labs has suggested that IFN-?, while clearly important, may not be the only factor required for macrophage-based control of infection in vivo. Indeed, several groups have reported that CD4 T cells can also mediate IFN-? independent control of infection in vivo. We have developed an ex vivo culture system that recapitulates CD4 T cell dependent but IFN-? independent control of infection, which provides a model system for mechanistic studies. Intriguingly, our preliminary data suggest that macrophages activated by IFN-? independent mechanisms activate aerobic glycolysis and HIF-1? without producing NO. Finally, very little is known about how macrophages support large scale changes in metabolism via regulated metabolite transport. Here we proposed to further our understanding of both IFN-? dependent and independent macrophage based control of Mtb infection in three aims: 1) Determine the importance of NO/HIF-1? for cell intrinsic control of Mtb infection in vivo 2) Demonstrate that IFN-? independent control of Mtb infection requires aerobic glycolysis and HIF-1? 3) Demonstrate that solute carrier proteins play a role in regulating HIF-1? dependent control of infection by supporting metabolite transport across cell membranes.